Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein;dynamic studies at the molecular level are almost impossible. In this study, we introduce a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and t-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and t-tubules, and analyzed insulin-stimulated exocytosis at the single vesicle level by TIRF and confocal microscopy. We found that only 10% of intracellular GLUT4 pool comprised mobile vesicles, while most of the GLUT4 structures remained stationary or tethered at the sarcolemma or t-tubules. In fact, most of insulin-stimulated exocytosis emanated from pre35 tethered vesicles, while the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action, but rather locally distributed, most likely, pre-tethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis.